Altered FAD2 and FAD3 genes in Brassica and the molecular marker-assisted detection thereof

ABSTRACT

The present invention provides methods of marker-assisted selection for high oleic/low linolenic traits in canola and in other oil seed crop species, as well as isolated nucleic acids for use as molecular markers in such methods. In particular, molecular markers and  Brassica  nucleic acid corresponding to fad2 and fad3 gene mutations are disclosed. The markers of the present invention are highly useful for the direct selection of desirable fad2 and fad3 alleles during marker-assisted trait introgression and breeding. In one aspect of the embodiment, two single nucleotide polymorphism (SNP) markers are provided that correspond to the alleles. Thus, the present invention advantageously permits one of skill in the art to breed for the molecular markers described herein, or derivatives thereof, rather than breeding for a high oleic/low linolenic phenotype.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a national phase entry under 35 U.S.C. §371 ofInternational Patent Application PCT/US2004/003852, filed Feb. 11, 2004,published in English as International Patent Publication No. WO2004/072259 on Aug. 26, 2004, which claims priority to U.S. PatentApplication No. 60/446,429 filed Feb. 11, 2003, the entire contents ofwhich is hereby incorporated herein by this reference.

TECHNICAL FIELD

The present invention relates generally to methods and materials for usein plant breeding. More specifically, the present invention relates tothe marker-assisted identification of genes encoding phenotypic traitsin oil seed plant species, and in Brassica species in particular.

BACKGROUND

The genus Brassica includes canola, one of the world's most importantoilseed crops, and the most important oilseed crop grown in temperategeographies. Canola has been traditionally characterized as Brassicanapus (a species derived as a result of inter-specific crosses ofBrassica rapa and Brassica oleracea) in which erucic acid andglucosinolates have been eliminated or significantly reduced throughconventional breeding. The majority of canola oil is in the form ofvegetable oils produced for human consumption. There is also a growingmarket for the use of canola oil in industrial applications.

Canola is a polyploid species considered to have arisen from thehybridization of Brassica oleracea, having a diploid C genome, andBrassica rapa, having a diploid A genome. Cytogenetic investigationrevealed the AA and CC genomes show a degree of relatedness, beingpartially homologous to one another and thought to have been derivedfrom a common ancestor genome (Prakash and Hinata, 1980). Althoughtechnically classified as diploids, the genomes of both progenitorspecies contain a high percentage of regions duplicative of one another(Song et al., 1991). Genetic analysis revealed that the AA genome ofBrassica rapa contributed ten chromosomes to Brassica napus, whileBrassica oleracea contributed nine chromosomes from its CC genome as thematernal donor (Song et al., 1992).

The quality of edible and industrial oil derived from a particularvariety of canola seed is determined by its constituent fatty acids, asthe type and amount of fatty acid unsaturation have implications forboth dietary and industrial applications. Conventional canola oilcontains about 60% oleic acid (C18:1), 20% linoleic acid (C18:2) and 10%linolenic acid (18:3). The levels of polyunsaturated linolenic acidtypical of conventional canola are undesirable as the oil is easilyoxidized, the rate of oxidation being affected by several factors,including the presence of oxygen, exposure to light and heat, and thepresence of native or added antioxidants and pro-oxidants in the oil.Oxidation causes off-flavors and rancidity as a result of repeatedfrying (induced oxidation) or storage for a prolonged period(auto-oxidation). Oxidation may also alter the lubricative and viscousproperties of canola oil.

Oils exhibiting reduced levels of polyunsaturated fatty acids andincreases in the level of monounsaturated oleic acid relative toconventional canola oil are associated with higher oxidative stability.The susceptibility of individual fatty acids to oxidation is dependenton their degree of unsaturation. Thus, the rate of oxidation oflinolenic acid, which possesses three carbon-carbon double bonds, is 25times that of oleic acid, which has only one double bond, and two timesthat of linoleic acid, which has two double bonds. Linoleic andlinolenic acids also have the most impact on flavor and odor becausethey readily form hydroperoxides. High oleic oil (≧70% oleic) is lesssusceptible to oxidation during storage, frying and refining, and can beheated to a higher temperature without smoking, making it more suitableas cooking oil.

Two strategies are generally used to increase the oxidative stability ofcanola oil. In one approach, partial hydrogenation is used to lowerlinolenic acid content. Unfortunately, partial hydrogenation leads tothe formation of trans-fatty acids, which have been linked to elevatedlevels of low-density lipoprotein cholesterol (LDL or “bad” cholesterol)in the blood, and consequently, to an increased risk of coronary heartdisease. The second major strategy involves breeding programs to developcanola varieties with high oleic and low linolenic acid levels relativeto conventional canola oil. High oleic and low linolenic mutants havebeen produced through mutagenesis (Rakow, 1973; Wong et al., 1991; Auldet al., 1992) and transgenic modification (Debonte and Hitz, 1996).Examples of commercially sold canola varieties having a fatty acidprofile of C18:1 above 70% and C18:3 below 3.5% are the Nexera™varieties, marketed by Dow AgroSciences LLC (Indianapolis, Ind.), whichvarieties produce Natreon™ oil. One such line, AG019 (a Nexera™ variety)contains 71% to 78% oleic (C18:1) and <3% linolenic (C18:3) acid. AG019was originally created by ethyl methanesulphonate (EMS) mutagenesis andis described in U.S. Pat. No. 6,169,190 B1 to Sernyk, assigned to theassignee of the present invention.

Current methods for producing F₁ hybrid Brassica seeds have definitelimitations in terms of cost and seed purity. Generally, these methodsrequire stable, sib-incompatible and self-incompatible, nearlyhomozygous parental breeding lines, which parental breeding lines areavailable only after repeated selfing to generate inbred lines.Furthermore, inbreeding to develop and maintain the parental lines isaccomplished by labor-intensive techniques, such as bud pollination,since Brassica hybrid seed production systems based on self-incompatibletraits must utilize strongly self-incompatible plants. Environmentalconditions during the breeding process, such as temperature andmoisture, typically affect plant lipid metabolism, thus also affectingthe content level of fatty acids (Harwood, 1999). Environmentalvariability, therefore, makes the phenotypic selection of plants lessreliable. Deng and Scarth (1998) found that increase in post-floweringtemperature significantly reduced the levels of C18:3 and increasedC18:1. Similar results were reported in other studies (Yermanos andGoodin, 1965; Canvin, 1965).

Breeding for low linolenic varieties is particularly challenging sinceC18:3 content is a multi-gene trait and inherited in a recessive mannerwith a relatively low heritability. Genetic analysis of a populationderived from the cross between “Stellar” (having a low C18:3 content(3%)) and “Drakkar” (having a “conventional” C18:3 level (9% to 10%))indicated that the low C18:3 trait was controlled by two major loci withadditive effects designated L1 and L2 (Jourdren et al., 1996b). Thesetwo major loci controlling C18:3 content were found to correspond to twofad3 (fatty acid desaturase 3) genes; one located on A genome(originating from Brassica rapa) and the other on the C genome(originating from Brassica oleracea) (Jourdren et al., 1996; Barret etal., 1999).

Traits that are continuously varying due to genetic (additive,dominance, and epistatic) and environmental influences are commonlyreferred to as “quantitative traits.” Quantitative traits may bedistinguished from “qualitative” or “discrete” traits on the basis oftwo factors: environmental influences on gene expression that produce acontinuous distribution of phenotypes; and the complex segregationpattern produced by multigenic inheritance. The identification of one ormore regions of the genome linked to the expression of a quantitativetrait led to the discovery of Quantitative Trait Loci (“QTL”). Thormannet al. (1996) mapped two QTL that explained 60% of the variance for thelinolenic content, while Somers et al. (1998) identified three QTL thatcollectively explained 51% of the phenotypic variation of C18:3 content.A three-locus additive model was also reported by Chen and Beversdorf(1990). Rücker and Röbbelen (1996) indicated that several minor genesare most likely involved in the desaturation step.

Heritability for C18:3 content was estimated to be 26% to 59% (Kondraand Thomas, 1975) (where the variability of heritability is a functionof genetics as opposed to environmental factors). Complexity of theinheritance of linolenic acid may be due to the fact that linolenic acidcan be synthesized either from the desaturation of C18:2 or theelongation of C16:3 (Thompson, 1983).

In contrast to linolenic acid, inheritance of oleic acid is lesscomplex, and the heritability of oleic acid is relatively high. It isreported that high oleic acid content is controlled by a major locuscalled fad2 (fatty acid desaturase 2) gene which encodes the enzymeresponsible for the desaturation of oleic acid to linoleic acid (C18:2)(Tanhuanpaa et al., 1998; Schierholt et al., 2001). All of thefunctional gene copies of the fad2 gene that have been reported andmapped to date are located on the A-genome-originated linkage group N5(Scheffler et al., 1997; Schierholt et al., 2000). Chen and Beversdorf(1990) reported that the accumulation of oleic acid was controlled by atleast two segregation genetic systems, one acting on chain elongationand the other involving desaturation. Heritability for C18:1 content wasestimated to be 53% to 78% (Kondra and Thomas 1975) and 94% (Schierholtand Becker, 1999), respectively. Due to the higher heritability, theexpression of C18:1 content is environmentally less affected andrelatively stable (Schierholt and Becker, 1999).

In Nexera™ canola germplasm, one to two genes are found to control C18:1content and at least three genes are involved in C18:3 expression. Insegregating progenies, the distribution of seed C18:3 content iscontinuous, thereby making it difficult to identify genotypic classeswith desirable C18:3 levels. In addition, there is a low correlation infatty acid content between greenhouse (GH) and field-grown plants,further making it challenging to reliably select GH plants withdesirable levels of C18:3.

Molecular maker selection is based on genotypes and is, therefore,independent from environmental effects. Molecular markers wouldalleviate the problem of the unreliable selection of plants in thegreenhouse attributable to the low correlation in fatty acid contentbetween greenhouse-grown plants and field-grown plants. Significantly,molecular markers tightly linked to the genes controlling C18:1 andC18:3 content would allow early selection of plants carrying genes forhigh C18:1 and low C18:3. Marker-assisted selection at early stage willsignificantly save greenhouse space, therefore, improve the efficiencyof greenhouse use, and reduce the breeding workload in the field.

More generally, molecular markers have advantages over morphologicalmarkers in that: molecular markers can be highly polymorphic whilemorphological markers are strictly phenotype dependent; morphologicalmarkers may interfere in the scoring of certain quantitative phenotypeswhile molecular markers exhibit a 1:1 relationship between genotype andphenotype (thus allowing the unambiguous scoring of all possiblegenotypes for a given locus); and epistatic interactions tend to limitthe number of morphological markers useful in a population, whilemolecular markers do not interact epistatically.

Different types of molecular markers such as RAPD (random-amplifiedpolymorphic DNA) markers (Tanhuanpaa et al., 1995; Hu et al., 1995;Rajcan et al., 1999; Jourdren et al., 1996), RFLP (restriction fragmentlength polymorphism) markers (Thormann et al., 1996) and SCAR(sequence-characterized amplified region) markers (Hu et al., 1999) havebeen identified to be associated with low C18:3 levels in Brassicanapus. Molecular markers have also been identified for high C18:1content. A RAPD marker was identified to be linked to the QTL affectingoleic acid concentration in spring turnip rape (B. rapa ssp. oleifera)and was later converted into a SCAR marker (Tanhuanpaa et al., 1996).Schierholt et al. (2000) identified three AFLP markers linked to a higholeic acid mutation in winter oilseed rape (B. napus L.). Tanhuanpaa etal. (1998) developed an allele-specific PCR marker for oleic acid bycomparing the wild-type and high-oleic allele of the fad2 gene locus inspring turnip rape (B. rapa ssp. oleifera). However, most of thesemarkers are low-throughput markers, such as RAPD, AFLP and RFLP, and arenot suitable for large-scale screening through automation.

Therefore, what is needed in the art are molecular markers suitable foridentifying canola plants producing a seed oil with desired levels ofhigh oleic and low linolenic acid, which render the oil sufficientlystable for uses in various dietary and industrial applications. It wouldbe further advantageous to map genes responsible for oleic and linolenicacid concentration and to develop high throughput PCR markers linked tohigh oleic and low linolenic acid content in order to facilitate theselection of these traits in oil seed crop trait introgression andbreeding.

SUMMARY OF THE INVENTION

The present invention provides methods of marker-assisted selection incanola and other oil seed crop species, as well as isolated nucleicacids for use as molecular markers in such methods.

In a first embodiment, an isolated and purified genetic markerassociated with high oleic oil content in Brassica is provided. Themarker maps to a linkage group selected from the group consisting of N5and N1 in the Brassica genome, and has the sequence of SEQ ID NO. 5 or aderivative thereof. An isolated and purified genetic marker associatedwith low linolenic oil content in Brassica is also provided. The markermaps to a linkage group selected from the group consisting of N14 and N4in the Brassica genome, and has the sequence of SEQ ID NO. 6 or aderivative thereof.

In another embodiment, the present invention provides nucleic acidsequences corresponding to mutated fad2 and/or fad3 genes. These genesmay be introduced into canola or other oil seed plants by any of anumber of known methods in the art. Additionally, wild-type fad2 and/orfad3 may be altered by known in vivo or in vitro methods to correspondto the fad2 and/or fad3 genes of the present invention.

In an additional embodiment, molecular markers corresponding to the fad2and fad3 gene mutations are disclosed. The markers of the presentinvention are highly useful for the direct selection of desirable fad2and fad3 alleles during marker-assisted trait introgression andbreeding. In one aspect of the embodiment, two single nucleotidepolymorphism (SNP) markers are provided that correspond to the alleles.Thus, the present invention advantageously permits one of skill in theart to breed for the molecular markers described herein, or derivativesthereof, rather than breeding for the phenotype. In a relatedembodiment, methods of marker-assisted selection for high oleic and/orlow linolenic oil seed plants are disclosed. In one aspect of theembodiment, methods for reliably and predictably introgressing traitsfor high oleic and/or low linolenic acid content into Brassica germplasmare provided. The methods include: (a) identifying one or more nucleicacid markers for marker-assisted selection among Brassica lines to beused in a Brassica breeding program, wherein the nucleic acid markersmap to at least one of linkage groups N14, N4, N5 or NI and wherein thenucleic acid markers are selected from among any of SEQ. ID. NOs. 5 and6, and (b) introgressing the traits for high oleic and/or low linolenicacid content into Brassica by performing marker-assisted selection. Afurther method is drawn to identifying nucleic acid as a modulator ofhigh oleic and/or low linolenic acid content in Brassica, and comprisesexposing Brassica nucleic acid to a molecular marker selected from thegroup consisting of SEQ. ID. Nos. 5 and/or 6.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows partial genomic nucleotide sequences of the fad2 genecloned from DMS100 and Quantum. The top is DMS100 sequence (SEQ. ID. NO.7) and bottom is Quantum sequence (SEQ. ID. NO. 9). The arrowheadindicates a single nucleotide mutation of C to T, which resulted in astop codon (TAG) (shaded). The forward and reverse primers for PCR-basedmutant allele-specific marker are bolded and underlined.

FIG. 2 provides amino acid sequences of the fad2 gene, degenerated fromthe genomic nucleotide sequence cloned from DMS100 (SEQ. ID. NO. 8),Quantum (SEQ. ID. NO. 10) and from a published Brassica napus fad2 gene(BNfad2) (SEQ. ID. NO. 11). The arrowhead indicates the position of thestop codon resulting from a single nucleotide mutation (C to T) inDMS100.

FIG. 3 shows genomic nucleotide sequences of the fad3c gene cloned fromDMS100 and Quantum. The top is the DMS100 sequence (SEQ. ID. NO. 12) andthe bottom is the Quantum sequence (SEQ. ID. NO. 13). Exons are boxed,introns are unboxed, which correspond to exons 4, 5, 6 and 7 and introns4, 5 and 6 of the fad3 gene in Brassica rapa and Arabidopsis. Thearrowhead indicates a single nucleotide mutation of G to A. The forwardand reverse primers for PCR-based mutant allele-specific markers arebolded and underlined.

FIG. 4 provides a table that correlates the mutant allele-specificmarkers and fatty acid content of 184 DH lines derived from the cross ofQuantum and DMS100, as well as electrophoresis results of PCR productsamplified from the mutant allele-specific marker for the fad2 gene.

FIG. 5 is a QTL map of showing one major (N5) and one minor (N1) QTLregion for high C18:1, and three QTL regions (N4 and N14) for low C18:3detected by markers of the present invention.

FIG. 6 provides Invader Assay results for mutant and wild-type allelesof fad2 and fad3 genes.

FIGS. 7 and 8 provide a list of the various PCR markers developed andtested for high oleic and/or low linolenic trait identification.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates generally to methods and materials for usein plant breeding. In a preferred embodiment, the present inventionrelates to methods and compositions of matter for marker-assistedidentification of genes encoding high oleic, low linolenic traits incanola.

By “genetic locus” is meant a location on a chromosome.

By “genomic locus” is meant a location within the entire set ofchromosomes of an organism.

As used herein, “linkage disequilibrium” refers to a statisticalassociation between two loci or between a trait and a marker.

As used herein, “marker” includes reference to a locus on a chromosomethat serves to identify a unique position on the chromosome. A genotypemay be defined by use of one or a plurality of markers.

The term “derivative,” as used herein, refers to a modification of asequence disclosed in the present invention. Illustrative of suchmodifications with regard to molecular markers would be thesubstitution, insertion, and/or deletion of one or more bases relatingto a nucleic acid sequence of a marker disclosed herein that preserve,slightly alter, or increase the function of the molecular marker inidentifying one or more high oleic and/or low linolenic traits inBrassica or other oil seed crop species. Such derivatives can be readilydetermined by one skilled in the art, for example, using computermodeling techniques for predicting and optimizing sequence structure.The term “derivative” thus also includes nucleic acid sequences havingsubstantial sequence homology with the disclosed marker sequences hereinsuch that they are able to have the disclosed functionalities for use inmarker-assisted breeding.

The term “homology,” as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence that lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

The terms “identity” and “similarity,” as used herein and as known inthe art, are relationships between two polypeptide sequences or twopolynucleotide sequences, as determined by comparing the sequences. Inthe art, identity also means the degree of sequence relatedness betweentwo polypeptide or two polynucleotide sequences as determined by thematch between two strings of such sequences. Both identity andsimilarity can be readily calculated (Computational Molecular Biology,A. M. Lesk, ed., Oxford University Press, New York (1988); Biocomputing:Informatics and Genome Projects, D. W. Smith, ed., Academic Press, NewYork (1993); Computer Analysis of Sequence Data, Part I, A. M. Griffinand H. G. Griffin, eds., Humana Press, New Jersey (1994); SequenceAnalysis in Molecular Biology, G. von Heinje, Academic Press (1987); andSequence Analysis Primer, M. Gribskov and J. Devereux, eds., StocktonPress, New York (1991)). Methods commonly employed to determine identityor similarity between two sequences include, but are not limited tothose disclosed in H. Carillo and D. Lipman, SIAM J. Applied Math.48:1073 (1988). Preferred methods to determine identity are designed togive the largest match between the two sequences tested. Methods todetermine identity and similarity are codified in computer programs.Typical computer program methods to determine identity and similaritybetween two sequences include: GCG program package (J. Devereux et al.,Nucleic Acids Research 12 (1):387 (1984)), BLASTP, BLASTN, FASTA andTFASTA (S. F. Atschul et al., J. Mol. Biol. 215:403 (1990)).

An “insertion” or “addition,” as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

The term “statistically associated” refers to the tendency of two eventsto occur together at a frequency greater than that attributable tochance, where the frequency attributable to chance is represented by apredetermined level of significance. Statistical association can bedetermined by any one of a number of significance tests well known tothose in the art, for example, ANOVA or t-tests. See, e.g., StatisticalMethods, G. W. Snedecor and W. G. Cochran, Iowa State University Press,Ames, Iowa (1985). Significance levels for alpha are preferably lessthan 0.01. For example, levels of significance for this invention couldrange between 0 and about 0.250, e.g., less than about 0.0001, 0.00050,0.0010, 0.0050, 0.010, 0.025, 0.050, 0.100, or 0.250.

The term “stringency” is used herein to describe the conditions oftemperature, ionic strength, and the presence of other compounds such asorganic solvents, under which nucleic acid hybridizations are conducted.Those skilled in the art will recognize that “stringency” conditions maybe altered by varying the parameters just described, either individuallyor in concert. With “high stringency” conditions, nucleic acid basepairing will occur only between nucleic acid fragments that have a highfrequency of complementary base sequences (for example, hybridizationunder “high stringency” conditions may occur between homologs with about85% to 100% identity, preferably about 70% to 100% identity). Withmedium stringency conditions, nucleic acid base pairing will occurbetween nucleic acids with an intermediate frequency of complementarybase sequences (for example, hybridization under “medium stringency”conditions may occur between homologs with about 50% to 70% identity).Thus, conditions of “weak” or “low” stringency are often required withnucleic acids that are derived from organisms that are geneticallydiverse, as the frequency of complementary sequences is usually less.

As used in the present application, the term “substantial sequencehomology” is used to indicate that a nucleotide sequence (in the case ofDNA or RNA) or an amino acid sequence (in the case of a protein orpolypeptide) exhibits substantial, functional or structural equivalencewith another nucleotide or amino acid sequence. Any functional orstructural differences between sequences having substantial sequencehomology will be de minimis; that is, they will not affect the abilityof the sequence to function as indicated in the present application.Sequences that have substantial sequence homology with the sequencesdisclosed herein are usually variants of the disclosed sequence, such asmutations, but may also be synthetic sequences.

A “substitution,” as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

Canola varieties DMS100 (mutant type) and Quantum (wild type) were usedin the cloning of fad2 (fatty acid desaturase-2) and fad3 (fatty aciddesaturase-3) alleles. The variety DMS100 was derived from an F₄ bulk ofa single F₃ plant selection originating from the cross of Global×AG019sister line. DMS100 is a HOLL (High Oleic and Low Linolenic) line witholeic acid content at about 77% and linolenic acid content at about 3%.Quantum is a commercial variety and contains low oleic acid (˜66%) andhigh linolenic acid (˜7%) content. As discussed in detail herein,sequencing of DMS100 genomic clones of fad2 and fad3 desaturase enzymesinvolved in the fatty acid synthesis pathway revealed single nucleotidemutations in each of the genes. Further sequence analyses show themutations to be the cause of altered fatty acid contents in DMS100.These two mutations are distinct from previously published mutations(Tanhuanpää et al., 1998; Jourdren, 1996), and the use of thesesequences as isolated nucleic acid conferring HOLL traits is an aspectof the present invention.

C18:1 content in canola is influenced by a fad2 gene that encodes anenzyme (endoplasmic delta 12 oleate desaturase) responsible for thedesaturation of oleic acid (C18:1) to linoleic acid (C18:2). In theExamples that follow, nine DMS100 clones and ten Quantum clones weresequenced. The sequence analysis and alignment of these clonesidentified a single nucleotide mutation, C to T, at position 411 thatconsistently occurred in the fad2 gene sequence of all the DMS100 clones(SEQ. ID. NO. 7), but not the Quantum clones (SEQ. ID. NO. 9) (see FIG.1). Further analysis indicated that this single nucleotide mutationoccurred in the coding sequence (exon) of the fad2 gene (see FIG. 2). Asfurther shown in FIG. 1, the mutation of C to T created a stop codon(TAG) that causes early termination of the polypeptide chain duringtranslation. The stop codon results in the incorporation of only 185amino acids into the polypeptide instead of all 384 amino acids of thefull-length polypeptide (FIG. 2). The truncated polypeptide appears tohave little, if any, function as an active desaturase for thedesaturation of oleic acid to linoleic acid, thus leading to theaccumulation of oleic acids in the seeds of the mutant line. The lack offunctionality of the mutant fad2 gene explains the significantly higherC18:1 content (77%) of DMS100 relative to the wild-type line Quantum(66%).

The fad3 gene encodes for endoplasmic delta-15 linoleic desaturase, anenzyme responsible for the desaturation of linoleic acid (C18:2) tolinolenic acid (C18:3). Two fad3 genes (fad31 and fad32) in particularhave been reported to control linolenic content. Seven DMS100 clones andsix Quantum clones of fad31 and six DMS100 clones and six Quantum clonesof fad32 were sequenced. Sequence analysis and alignment revealed nosequence difference between DMS100 and Quantum clones for fad31 (datanot shown). However, sequence alignment revealed a single nucleotidemutation, G to A, at the first base of 5′ splice site of the thirdintron in fad32 gene (see FIG. 3). This intron corresponds to the intron6 of the fad3 gene in B. rapa (Tanhuanpää, 2000) and Arabidopsis(Nishiuchi et al., 1994). The fad3 gene of B. rapa and Arabidopsiscontains eight exons and seven introns, while the sequence examinedcovers exons 4 (partial), 5, 6 and 7 (partial) and introns 4, 5 and 6.This interpretation of exons/introns in the fad3 gene is supported bythe fact that the fad3 gene sequence is highly conserved among sequencedBrassica species and Arabidopsis.

Plant introns contain highly conserved 5′ splice sites (exon/intronjunction—AG/GTAAG) and 3′ splice sites (intron/exon junction—TGCAG/G.The first two nucleotides in the 5′ splice site intron junctionsequence, +1G and +2T, have shown 100% and 99% conservation,respectively, among over 1000 Arabidopsis introns studied (Lorkovic,2000; and Brown, 1996). The accuracy of splicing depends on themechanisms of intron signal recognition and the correct selection of 5′and 3′ splice sites. Referring again to FIG. 3, the mutation of +1G to+1A at the 5′ splice site (position 530) identified herein can abolishsplicing or lead to exon skipping, i.e., the affected exon (exon 6) andboth flanking introns are removed in a single splicing event (Lorkovicet al., 2000; Simpson et al., 1998). Such exon skipping could lead tosynthesis of a polypeptide missing the amino acids encoded by the exon 6of the fad3 gene. The mutation could also block splicing at the normal5′ splice site and activate cryptic splice sites at different positions,which can cause cryptic splicing of the affected exon together with thedownstream intron (McCullough et al., 1993). Such cryptic splicing couldlead early termination of translation and synthesis of a shorterpolypeptide for delta-15 linoleate desaturase encoded by fad3. This willoccur because the intron contains stop codons in all three possiblereading frames and hence, exons 7 and 8 will remain untranslated. Theincomplete translation of the fad3 can inactivate the enzyme and blockthe desaturation of linoleic acid (C18:2) to linolenic acid (C18:3),resulting in the decrease of C18:3 accumulation in canola seeds.

These data strongly suggest that the single nucleotide mutationsidentified in the fad2 and fad3 genes are factors that account for theincrease in oleic acid and decrease in linolenic acid contents in thecanola line DMS100. Using the molecular markers of the present inventionor markers with substantial homology thereto, these two mutations mayserve to allow marker-assisted introgression into canola lines makinguse of DMS100, its progeny or derivatives, or transgenic versions of itsmutated fad2 and fad3 genes (SEQ. ID. NO. 7 (see FIG. 1) and SEQ. ID.NO. 12 (see FIG. 3), respectively), for purposes of developing HOLLcanola.

Identification of Mutations in fad2 and fad3 Genes

Referring to FIG. 1, primers homologous to the Arabidopsis fad2 genesequence were used to amplify genomic DNA fragments of the fad2 genefrom B. napus lines DMS100 and Quantum. The primer pair FAD2-2F:CAATCCCTCGCTCTTTCTCCTACC (SEQ. ID. NO. 1) and FAD2-6R:CCTTTCTTGTCACCTTCCCTGTCC (SEQ. ID. NO. 2) amplified a fad2 fragment ofthe same length (986 bp) from each of the two parents. The amplifiedfragments were then cloned and sequenced to investigate the sequencedifferences of fad2 gene between the two parents.

Genomic DNA fragments corresponding to the fad31 and fad32 genes wereamplified from DMS100 and Quantum lines using PCR. The primers foramplification were designed from the published B. napus fad31 and fad32gene sequences (Brunel et al., 1999, GenBank Accession AF056569 andAF056570, respectively). The fad31 fragments amplified by the primerpairs BNFD31-CF (GAGGCTTGGACGACCACTTG) (SEQ. ID. NO. 3) and BNFD31-CR(GACTGGACCAACGAGGAATG) (SEQ. ID. NO. 4) and fad32 fragments amplified bythe primer pairs BNFD32-CF (CAAGAATTTGTCCCACAGTACAC) (SEQ. ID. NO. 14)and BNFD32-CR (CAACTGTTGTTAATCCTCCACG) (SEQ. ID. NO. 15) were clonedbecause these fragments covered more sequences of each gene. SevenDMS100 clones and six Quantum clones of fad31 and six DMS100 clones andsix Quantum clones of fad32 were sequenced. Sequence analysis andalignment revealed no sequence difference between DMS100 and Quantum forfad31 (data not shown). However, sequence alignment revealed a singlenucleotide mutation, G to A, at the first base of 5′ splice site of thethird intron in fad32 gene (see FIG. 3). This intron corresponds to theintron 6 of the fad3 gene in B. rapa (Tanhuanpää, 2000) and Arabidopsis(Nishiuchi et al., 1994). The fad3 gene of B. rapa and Arabidopsiscontains eight exons and seven introns, while the sequence examinedcovers exons 4 (partial), 5, 6 and 7 (partial) and introns 4, 5 and 6.This interpretation of exons/introns in the fad3 gene is supported bythe fact that the fad3 gene sequence is highly conserved among sequencedBrassica species and Arabidopsis.

Plant introns contain highly conserved 5′ splice sites (exon/intronjunction—AG/GTAAG) and 3′ splice sites (intron/exon junction—TGCAG/G.The first two nucleotides in the 5′ splice site intron junctionsequence, +1G and +2T, have shown 100% and 99% conservation,respectively, among over 1000 Arabidopsis introns studied (Lorkovic,2000; and Brown, 1996). The accuracy of splicing depends on themechanisms of intron signal recognition and the correct selection of 5′and 3′ splice sites. Referring again to FIG. 3, the mutation of +1G to+1A at the 5′ splice site (position 530) identified herein can abolishsplicing or lead to exon skipping, i.e., the affected exon (exon 6) andboth flanking introns are removed in a single splicing event (Lorkovicet al., 2000; Simpson et al., 1998). Such exon skipping could lead tosynthesis of a polypeptide missing the amino acids encoded by exon 6 ofthe fad3 gene. The mutation could also block splicing at the normal 5′splice site and activate cryptic splice sites at different positions,which can cause cryptic splicing of the affected exon, together with thedownstream intron (McCullough et al., 1993). Such cryptic splicing couldlead to early termination of translation and synthesis of a shorterpolypeptide for delta-15 linoleate desaturase encoded by fad3. This willoccur because the intron contains stop codons in all three possiblereading frames and hence, exons 7 and 8 will remain untranslated. Theincomplete translation of the fad3 can inactivate the enzyme and blockthe desaturation of linoleic acid (C18:2) to linolenic acid (C18:3),resulting in the decrease of C18:3 accumulation in canola seeds.

These data strongly suggest that the single nucleotide mutationsidentified in the fad2 and fad3 genes are factors that account for theincrease in oleic acid and decrease in linolenic acid contents in thecanola line DMS100. As shown in FIGS. 1 and 3, respectively,mutant-specific primers FAD2GM (CGCACCGTGATGGTTAACGGTTT) (SEQ. ID. NO.5) and FAD3cGM (ATAAATAATGTTGATCTACTTAT) (SEQ. ID. NO. 6) were designedfor purposes of detecting the mutant HOLL alleles of fad2 and fad32using PCR amplification. Using the molecular markers of the presentinvention, these two mutations may serve to allow marker-assistedintrogression into Brassica lines using the HOLL alleles of DMS100, itsprogeny or derivatives, or transgenic versions of its mutated fad2 andfad3 genes (SEQ. ID. NO. 7 (FIG. 1) and SEQ. ID. NO. 8 (FIG. 3),respectively) for purposes of developing HOLL canola.

Development of Mutant Allele-Specific SNP Markers for fad2 and fad3Genes

In a presently preferred embodiment, the single nucleotide mutationspresent in the fad2 and fad3 genes are used as SNP markers to tag thefad2 and fad3 genes for selection of high C18:1 and low C18:3 in canolabreeding. Mutant-specific primers (FAD2GM: CGCACCGTGATGGTTAACGGTTT (SEQ.ID. NO. 5); and FAD3cGM: ATAAATAATGTTGATCTACTTAT (SEQ. ID. NO. 6)) weredesigned in order to detect mutant alleles of fad2 and fad32 using PCRamplification. The primers were designed such that the mutated base(SNP) was at the 3′ end of one of the primers for allele-specific PCRamplification (FIGS. 1 and 3). Lists of the various PCR markersdeveloped and tested for HO/LL trait identification are provided inFIGS. 7 and 8. The primers specific to fad2 amplified a polymorphic bandthat was present in DMS100 and DNA bulks for high oleic acid (C18:1),but were absent in Quantum and the DNA bulks for low oleic acid (FIG.4).

This gene-specific marker was tested on a doubled haploid (DH)population derived from the cross of Quantum and DMS100, where it wasfound that the allele distribution was highly correlated to high C18:1(see FIG. 4, Table 1). The fad3 allele-specific primers also amplified apolymorphic fragment that was present in DMS100 but absent in Quantum.The analysis with the DH population indicated that this allele-specificmarker was statistically associated with low C18:3 (see FIG. 4, Table1). Thus, two gene-specific PCR-based markers that directly tag fad2 andfad3 gene mutations were successfully developed. Given the disclosure,variations or derivatives of the markers disclosed herein (includingmarkers of various types) based for example, on substantial homologyover a sufficient number of base pairs, will be readily apparent to oneof skill in the art.

Through genetic and QTL mapping using the DH population derived from thecross of Quantum×DMS100, one major (N5) and one minor (N1) QTL regionfor high C18:1, and three QTL regions (N4 and N14) for low C18:3 havebeen found (FIG. 5). This QTL mapping result is consistent with thegenetic analysis that high C18:1 is controlled by one major gene and lowC18:3 is controlled by multiple genes. The fad2 gene-based marker waslocated exactly at the mapped location of the major QTL locus for C18:1,supporting the fact that this QTL corresponds to the functional fad2gene that is affected by the mutation in DMS100. This is also consistentwith the previous studies that the fad2 gene is located on the linkagegroup N5 (Schierholt, 2000). The location of the fad3 gene-based markermatches exactly with the mapped location of one of the major QTL locifor C18:3 on the linkage group 14 (C genome), supporting the conclusionthat this QTL is the fad3c (fad3 in the C genome, previously calledfad32) gene and it is also affected by the second mutation in DMS100.

For molecular marker methods, see generally, The DNA Revolution byAndrew H. Paterson 1996 (Chapter 2) in: Genome Mapping in Plants (ed.Andrew H. Paterson) by Academic Press/R. G. Landis Company, Austin,Tex., pp. 7-21.

All publications, patents, and patent applications cited herein arehereby incorporated by reference. Unless otherwise noted herein,standard methods of DNA purification, restriction enzyme digestion,agarose gel analysis, DNA fragment isolation, ligation andtransformation may be used for purposes of the present invention. Suchmethods are described in, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2ded., 1989), and Ausubel et al., Current Protocols in Molecular Biology(New York: John Wiley and Sons) (1987), both of which are alsoincorporated by reference herein.

The present invention has of necessity been discussed herein byreference to certain specific methods and materials. The enumeration ofthese methods and materials was merely illustrative, and in no wayconstitutes any limitation on the scope of the present invention. It isto be expected that those skilled in the art may discern and practicevariations of or alternatives to the specific teachings provided herein,without departing from the scope of the present invention.

EXAMPLES Example 1 Plant Material

Canola varieties DMS100 (mutant type) and Quantum (wild type) were usedin this study for cloning of fad2 (fatty acid desaturase-2) and fad3(fatty acid desaturase-3) alleles. DMS100 is a HOLL (High Oleic and LowLinolenic) line with oleic acid content at about 77% and linolenic acidcontent at about 3%. It is derived from an F4 bulk of a single F3 plantselection originating from the cross of Global×AG019 sister line.Quantum is a commercial variety and contains low oleic acid (˜66%) andhigh linolenic acid (˜7%) content. A double haploid (DH) population wasdeveloped by microspore culture from F1 plants of the cross betweencanola line Quantum and DMS100. The DH population comprised of 604lines. A complete fatty acid analysis of the seeds of the DH lines andtheir parents was implemented by using gas chromatography. Of the 604 DHlines, 183 were randomly selected for marker analysis and mapping.

Example 2 Genomic DNA Extraction and Quantification

DNA of both parental lines and 183 DH lines was extracted from theleaves of two-week-old greenhouse-grown plants using Qiagen DNeasy 96Plant Test Kit. The details of DNA extraction procedures are describedin the DNeasy™ 96 Plant Test Kit Handbook. This kit allowed DNA to beextracted in a 96-well format for a high throughput extraction.

For DNA quantification, PicoGreen dye was diluted 200-fold into 1×TEbuffer. In a microtiter plate, 100 μl of the diluted PicoGreen dyesolution were added into each well and then 5 μl of each DNA sample orDNA standards (5 μg/ml, 10 μg/ml and 20 μg/ml) were added. The plate wasthen agitated on a plate shaker briefly and read using the Spectra MaxGEMINIS XS microplate fluorometer from Molecular Devices.

Example 3 PCR Amplification

PCR amplification reactions contained 20 to 30 ng of genomic DNA, 0.25μM 10-mer primer, 2.5 mM MgCl₂, 0.2 mM of each dNTP, 1×PCR buffer and0.6 units of Tag DNA polymerase. Amplifications were performed in aGeneAmp PCR System 9700 programmed for 35 cycles of 45 seconds at 94°C., 30 seconds at 55° C. to 60° C., 1 minute at 72° C. and ending with 7minutes at 72° C.

Example 4 Cloning of fad2 and fad3 Alleles

The fad2 fragments of parental lines DMS100 and wild-type line Quantumwere amplified by using the primers homologous to Arabidopsis or B. rapafad2 gene sequences (Tanhuanpää et al., 1998). The fad2 fragmentsamplified from each of the parents by the primers FAD2-2F and FAD2-6Rwere cloned and sequenced. The primers FAD2-2F and FAD2-6R correspond tothe primers 2 and 6 of Tanhuanpää et al. (1998), respectively. Thesequences of these two primers are:

FAD2-2F: CAATCCCTCGCTCTTTCTCCTACC FAD2-6R: CCTTTCTTGTCACCTTCCCTGTCC

The DNA sequences of the fad31 and fad32 loci for C18:3 of B. napus weresearched and retrieved from GenBank. The GenBank accession number forfad31 and fad32 are AF056569 and AF066570, respectively. Three pairs ofprimers for each fad31 and fad32 locus were designed from fad31 andfad32 gene sequences by using Primer Express primer designing software(PE Applied Biosystems, Foster City, Calif.). The fad31 fragmentsamplified by the primers BNFD31-CF and BNFD31-CR and the fad32 fragmentsamplified by the primers BNFD32-CF and BNFD32-CR from each of theparents were cloned and sequenced.

The PCR amplification products of interest were resolved by agarose-gelelectrophoresis, and the bands of interest were excised from the gel.The excised bands were placed in a microfuge tube containing sterilizedwater and heated for five minutes in boiling water. The dissolved DNAwas amplified by PCR with the corresponding primer pairs. The amplifiedproducts were ligated to PCR2.1-TOPO cloning vector using a TA-cloningkit (Invitrogen Corp., San Diego, (Calif.) per manufacturer'sinstructions. The ligated products were then transformed into competentcells and plated on LB-agar plates containing ampicillin or kanamycin,X-GAL and IPTG to enable white/blue selection. White colonies in thetransformation plates were picked and identification of the cloned PCRproducts were verified by a digest with the restriction enzyme EcoR I,which revealed the vector DNA fragment and the insert fragment of theexpected size. The positive clones containing the insert were sequencedby Sequetech Corporation (Mountain View, Calif.).

Example 5 Invader Assay

Invader Assay kits specific to fad2 and fad3 gene mutations weredeveloped through Third Wave Technologies (Madison, Wis.). Theconcentration of DNA samples for Invader Assay was normalized to 15ng/μl using QiaGen Bio-Robot 3000 (Valencia, Calif.). Invader Assay wasperformed in 96-well plates per manufacturer's instruction. In brief,DNA samples were denatured at 95° C. for ten minutes. Seven μl of thedenatured DNA (15 ng/μl) and 8 μl of reaction mix (3 μl oligo mix and 5μl of 24 mM MgCl₂) were added into each well of 96-well Invader Assayplates. Then, each reaction was overlaid with 15 μl of mineral oil andthe plates were incubated in the BioOven III from St. John Associates,Inc. (Beltsville, Md.) at 63° C. for four hours. The reaction plateswere read using the Spectra Max GEMINIS XS microplate fluorometer fromMolecular Devices for fluorescent signals. Percent signal overbackground for the mutant allele was divided by the percent signal forwild-type allele for each sample to calculate the ratio. The genotypesof the samples were determined based on the calculated ratio. Resultsare provided in FIG. 6.

Example 6 Sequence and Data Analyses

The sequences were analyzed and aligned by using SeqWeb (version 2)web-based sequence analysis software in GCG software package (WisconsinUniversity). Linkage association between the markers and high oleic orlow linolenic (HO/LL) traits were determined by t-test analysis. Thegenetic linkage map was generated with JoinMap V2.0 computer softwareusing a minimum LOD of 3.0. Map distance was converted to centiMorgansusing the Kosambi function. Putative QTL regions associated with theC18:1 and C18:3 were located by interval mapping using the MapQTL V 3.0software. A LOD score of 3.0 was used to identify regions potentiallyaffecting the two fatty acid traits.

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What is claimed is:
 1. A method for reliably and predictablyintrogressing traits for high oleic and/or low linolenic acid contentinto Brassica germplasm, said method comprising: identifying one or morenucleic acid markers that map to at least one of linkage groups N14, N4,N5 or N1, wherein the nucleic acid markers comprise a nucleotidesequence within a Fad2 gene that is capable of hybridizing to thenucleic acid molecule of SEQ ID NO:8 under high stringency conditions,but is not capable of hybridizing to the nucleic acid molecule of SEQ IDNO:7 under high stringency conditions; crossing a Brassica plantcomprising the identified one or more nucleic acid markers with anotherBrassica plant that does not comprise the identified one or moremarkers; and selecting progeny from the cross that comprise theidentified one or more nucleic acid markers.
 2. The method of claim 1,wherein Brassica is canola.
 3. A method for reliably and predictablyintrogressing traits for high oleic and/or low linolenic acid contentinto Brassica germplasm, said method comprising: identifying one or morenucleic acid markers that map to at least one of linkage groups N14, N4,N5 or N1, wherein the nucleic acid markers comprise a nucleotidesequence within a Fad3 gene that is capable of hybridizing to thenucleic acid molecule of SEQ ID NO:12 under high stringency conditions,but is not capable of hybridizing to the nucleic acid molecule of SEQ IDNO:13 under high stringency conditions; crossing a first Brassica plantcomprising the identified one or more nucleic acid markers with a secondBrassica plant that does not comprise the identified one or moremarkers; and selecting progeny from the cross that comprise theidentified one or more nucleic acid markers.
 4. The method of claim 3,wherein Brassica is canola.
 5. A method for reliably and predictablyintrogressing traits for high oleic and/or low linolenic acid contentinto Brassica germplasm, said method comprising: identifying nucleicacid markers that map to at least one of linkage groups N14, N4, N5 orN1, wherein the nucleic acid markers comprise a nucleic acid markercomprising a nucleotide sequence within a Fad2 gene that is capable ofhybridizing to the nucleic acid molecule of SEQ ID NO:8 under highstringency conditions, but is not capable of hybridizing to the nucleicacid molecule of SEQ ID NO:7 under high stringency conditions, and anucleic acid marker comprising a nucleotide sequence within a Fad3 genethat is capable of hybridizing to the nucleic acid molecule of SEQ IDNO:12 under high stringency conditions, but is not capable ofhybridizing to the nucleic acid molecule of SEQ ID NO:13 under highstringency conditions; crossing a Brassica plant comprising theidentified markers with another Brassica plant that does not comprisethe identified nucleic acid markers; and selecting progeny from thecross that comprise the identified nucleic acid markers.
 6. The methodof claim 5, wherein Brassica is canola.
 7. A method for reliably andpredictably introgressing traits for high oleic and/or low linolenicacid content into Brassica germplasm, said method comprising:identifying one or more nucleic acid markers that map to at least one oflinkage groups N14, N4, N5 or N1, wherein the nucleic acid markerscomprise SEQ ID NO:5; crossing a first Brassica plant comprising theidentified one or more nucleic acid markers with a second Brassicaplant; and selecting progeny from the cross that comprise the identifiedone or more nucleic acid markers.
 8. The method according to claim 7,wherein Brassica is canola.
 9. The method according to claim 7, whereinthe one or more nucleic acid markers consist of a nucleic acid markerconsisting of the polynucleotide of SEQ ID NO:5, wherein the presence ofthe nucleic acid marker consisting of the polynucleotide of SEQ ID NO:5is determined in the first Brassica plant and the selected progeny bypolymerase chain reaction, and wherein the Brassica plants are canolaplants.
 10. A method for reliably and predictably introgressing traitsfor high oleic and/or low linolenic acid content into Brassicagermplasm, said method comprising: identifying one or more nucleic acidmarkers that map to at least one of linkage groups N14, N4, N5 or N1,wherein the nucleic acid markers comprise SEQ ID NO:6; crossing a firstBrassica plant comprising the identified one or more nucleic acidmarkers with a second Brassica plant, wherein the first Brassica plantcomprises a G to A mutation at the first base of the 5′ splice site ofthe third intron in the Fad3 gene; and selecting progeny from the crossthat comprise the identified one or more nucleic acid markers.
 11. Themethod according to claim 10, wherein Brassica is canola.
 12. The methodaccording to claim 10, wherein the one or more nucleic acid markersconsist of a nucleic acid marker consisting of the polynucleotide of SEQID NO:6, wherein the presence of the nucleic acid marker consisting ofthe polynucleotide of SEQ ID NO:6 is determined in the first Brassicaplant and the selected progeny by polymerase chain reaction, and whereinthe Brassica plants are canola plants.
 13. A method for reliably andpredictably introgressing traits for high oleic and/or low linolenicacid content into Brassica germplasm, said method comprising:identifying one or more nucleic acid markers that map to at least one oflinkage groups N14, N4, N5 or N1, wherein the nucleic acid markerscomprise a first nucleic acid marker comprising SEQ ID NO:5 and a secondnucleic acid marker comprising SEQ ID NO:6; crossing a first Brassicaplant comprising the identified one or more nucleic acid markers with asecond Brassica plant; and selecting progeny from the cross thatcomprise the identified one or more nucleic acid markers.
 14. The methodaccording to claim 13, wherein Brassica is canola.
 15. The methodaccording to claim 13, wherein the one or more nucleic acid markersconsist of two nucleic acid markers, the first nucleic acid markerconsisting of the polynucleotide of SEQ ID NO:5, and the second nucleicacid marker consisting of the polynucleotide of SEQ ID NO:6, wherein thepresence of the two nucleic acid markers is determined in the firstBrassica plant and the selected progeny by polymerase chain reaction,and wherein the Brassica plants are canola plants.